Selective adenosine receptor agents

ABSTRACT

Adenosine analogues which act selectively at adenosine receptors and which act in general as adenosine antagonists are disclosed. From in vitro studies it is known that specific physiological effects can be distinguished as a result of this selectivity and that adenosine receptor activity in vitro correlates with adenosine receptor activity in vivo. 
     Pharmaceutical preparations of the subject compounds can be prepared on the basis of the selective binding activity of the compounds disclosed herein which will enhance certain physiological effects while minimizing others, such as decreasing blood pressure without decreasing heart rate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 07/954,178, filed Sep. 30,1992, now U.S. Pat. No. 5,256,650, which is a continuation-in-part ofapplication Ser. No. 07/873,660, filed Apr. 22, 1992, now abandoned,which is a continuation of application Ser. No. 07/734,024, filed Jul.22, 1991, now abandoned, which is a continuation of application Ser. No.07/551,686, filed Jul. 9, 1990, now abandoned, which is a continuationof application Ser. No. 07/329,919, filed Mar. 29, 1989, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a group of compounds which areadenosine analogues and which act selectively at adenosine receptors.

BACKGROUND OF THE INVENTION

The profound hypotensive, sedative, antispasmodic, and vasodilatoryactions of adenosine were first recognized over 50 years ago.Subsequently, the number of biological roles proposed for adenosine haveincreased considerably. The adenosine receptors appear linked in manycells to adenylate cyclase. A variety of adenosine analogues have beenintroduced in recent years for the study of these receptor functions.Alkylxanthines, such as caffeine and theophylline, are the best knownantagonists of adenosine receptors.

Adenosine perhaps represents a general regulatory substance, since noparticular cell type or tissue appears uniquely responsible for itsformation. In this regard, adenosine is unlike various endocrinehormones. Nor is there any evidence for storage and release of adenosinefrom nerve or other cells. Thus, adenosine is unlike variousneurotransmitter substances.

Adenosine might be compared as a physiological regulator to theprostaglandins. In both cases the enzymes involved in the metabolicformation are ubiquitous and appear to be responsive to alterations inthe physiological state of the cell. Receptors for adenosine, like thosefor prostaglandins, are proving to be very widespread. Finally, bothprostaglandins and adenosine appear to be involved with the regulationof functions involving calcium ions. Prostaglandins, of course, derivefrom membrane precursors, while adenosine derives from cytosolicprecursors.

Although adenosine can affect a variety of physiological functions,particular attention has been directed over the years toward actionswhich might lead to clinical applications. Preeminent has been thecardiovascular effects of adenosine which lead to vasodilation andhypotension but which also lead to cardiac depression. Theantilipolytic, antithrombotic, and antispasmodic actions of adenosinehave also received some attention. Adenosine stimulates steroidogenesisin adrenal cells, again probably via activation of adenylate cyclase.Adenosine has inhibitory effects on neurotransmission and on spontaneousactivity of central neurons. Finally, the bronchoconstrictor action ofadenosine and its antagonism by xanthines represents an important areaof research.

It has now been recognized that there are not one but at least twoclasses of extracellular receptors involved in the action of adenosine.One of these has a high affinity for adenosine and, at least in somecells, couples to adenylate cyclase in an inhibitory manner. These havebeen termed by some as the A-1 receptors. The other class of receptorshas a lower affinity for adenosine and in many cell types couples toadenylate cyclase in a stimulatory manner. These have been termed theA-2 receptors.

Characterization of the adenosine receptors has now been possible with avariety of structural analogues. Adenosine analogues resistant tometabolism or uptake mechanisms have become available. These areparticularly valuable, since their apparent potencies will be lessaffected by metabolic removal from the effector system. The adenosineanalogues exhibit differing rank orders of potencies at A-1 and A-2adenosine receptors, providing a simple method of categorizing aphysiological response with respect to the nature of the adenosinereceptor. The blockade of adenosine receptors (antagonism) providesanother method of categorizing a response with respect to theinvolvement of adenosine receptors. It should be noted that thedevelopment of antagonists specific to A-1 or A-2 adenosine receptorswould represent a major breakthrough in this research field and in thepreparation of adenosine receptor selective pharmacological agentshaving specific physiological effects in animals.

SUMMARY OF THE INVENTION

The present invention relates to compounds having the following generalformula: ##STR1## wherein R₁ is hydrogen phenyl or β-D-ribofuranosyl; R₂is hydrogen, lower alkyl of from 1 to 4 carbon atoms or lower alkoxy offrom 1 to 4 carbon atoms; Y is --N═ or --C═; Z is --N═ or --C═, with theproviso that Y and Z are not identical; each X is independentlyhydrogen, hydroxy, lower alkyl of from 1 to 3 carbon atoms orhydroxyalkyl of from 1 to 3 carbon atoms; and n is an integer from 1 to3.

DETAILED DESCRIPTION OF THE INVENTION

The lower alkyl groups, as indicated above, contain 1 to 4 carbon atomsand this same definition applies to any use of the term below.Similarly, the lower alkoxy groups, as indicated above, contain 1 to 4carbon atoms and this definition applies to any use of the terms below.Examples of such alkoxy groups are methoxy, ethoxy, propoxy and butoxy.

Stereoisomerism is possible with the present compounds and the chemicalstructure as presented above is considered as encompassing all of thepossible stereoisomers and racemic mixtures of such stereoisomers. Morespecifically, when X in any of the --(CHX)_(n) -- as shown in Formula Iis other than hydrogen, chirality is exhibited about the respectivecarbon atom and optical isomerism is possible.

As examples of compounds of the present invention are the following:

1. (R)-β-[(9-phenyl-9H-purin-6-yl)amino]benzenepropanol

2. (S)-β-[(9-phenyl-9H-purin-6-yl)amino]benzenepropanol

3. (S)-β-[(2-propoxy-1H-purin-6-yl)amino]benzenepropanol

4. β-[(1H-purin-6-yl)amino]benzenepropanol

5.[S-(R*,S*)]-α-[1-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanol

6.[R-(S*,R*)]-α-[1-[(1-phenyl-6-propoxy-1H-pyrazolo-[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanol

7. β-[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanol

8.(R)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-amine

9.(S)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-amine

10. (S)-β-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]-pyrimidin-4-yl)amino]benzenepropanol

11. (R)-β-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]-pyrimidin-4-yl)amino]benzenepropanol

12.(R)-β-[(2-propoxy-9-β-D-ribofuranosyl-9H-purin-6-yl)amino]benzenepropanol

13.(S)-β-[(2-propoxy-9-β-D-ribofuranosyl-9H-purin-6-yl)amino]benzenepropanol

14. (R)-N-(1-phenylpropyl)-adenosine

15. ( S)-N-(1-phenylpropyl )-adenosine.

In general, the compounds of the present invention are formed byreacting, under appropriate conditions, a compound of the generalstructure: ##STR2## wherein Cl is chlorine; R₁ is hydrogen, phenyl orβ-D-ribofuranosyl; Y is --N═ or --CH═; and Z is --N═ or --CH═, with theproviso that Y and Z cannot be identical, with a compound of thestructure: ##STR3## wherein each X is independently hydrogen, hydroxy orlower hydroxyalkyl of from 1 to 3 carbon atoms, and n is an integer from1 to 3, to form a compound of the structure: ##STR4## wherein R₁ ishydrogen, phenyl or β-D-ribofuranosyl; Y is --N═ or --CH═; Z is --N═ or--CH═, with the proviso that Y and Z cannot be identical; X is hydrogen,hydroxy or a lower hydroxyalkyl of from 1 to 3 carbon atoms and n is aninteger from 1 to 3.

Likewise, compounds of the following general structure: ##STR5## can bemade by reacting a compound of the following general structure: ##STR6##with a selected alcohol of from 1 to 4 carbon atoms, such as n-propanol.

Therapeutic Utility Of Selective Adenosine Receptor Agents

The table below shows in more detail the potential therapeutic utilityof selective adenosine receptor agents in accordance with the presentinvention:

    ______________________________________                                                                     Receptor                                         Area        Effect           Correlate                                        ______________________________________                                        Cardiovascular                                                                            cardiotonic      A-1 antagonism                                   Cardiovascular                                                                            control tchycardia                                                                             A-1 agonism                                      Cardiovascular                                                                            increase coronary blood                                                                        A-2 agonism                                                  flow                                                              Cardiovascular                                                                            vasodilation     A-2 (atypical)                                                                agonism                                          Pulmonary   bronchodilation  A-1 antagonism                                   Pulmonary   mediation of autocoid                                                                          novel adenosine                                              release from mast                                                                              receptor inter-                                              cells, basophils action on cell                                                                surface                                          Pulmonary   stimulate respiration;                                                                         Ado antagonism                                               treat paradoxical ven-                                                        tilatory response                                                             (infants)                                                         Renal       inhibit renin release                                                                          A-1 agonism                                      Central Nervous                                                                           aid in opiate    Ado agonism                                      System      withdrawal                                                        Central Nervous                                                                           analgesic        A-1 agonism                                      System                                                                        Central Nervous                                                                           anticonvulsant   A-1 agonism                                      System                                                                        Central Nervous                                                                           antidepressant   A-1 agonism                                      System                                                                        Central Nervous                                                                           antipsychotic    Ado agonism                                      System                                                                        Central Nervous                                                                           anxiolytic       agonism                                          System                                                                        Central Nervous                                                                           inhibition of self-                                                                            Ado agonism                                      System      mutilation behavior                                                           (Lesch-Nyhan syndrome)                                            Central Nervous                                                                           sedative         A-2 agonism                                      System                                                                        ______________________________________                                    

In the cardiovascular, pulmonary and renal system targets, designedcompounds which are identified by receptor binding studies can beevaluated in functional in vivo tests which are directly indicative ofthe human physiological response. A good description of the pharmacologyand functional significance of purine receptors is presented by M.Williams in Ann. Rev. Pharmacol. Toxicol., 27, 31 (1987). In a sectionentitled "Therapeutic Targeting of Adenosine Receptor Modulators" it isstated that "adenosine agonists may be effective as antihypertensiveagents, in the treatment of opiate withdrawal, as modulators of immunecompetence and renin release, as antipsychotics and as hypnotics.Conversely, antagonists may be useful as central stimulants, inotropics,cardiotonics, antistress agents, antiasthmatics, and in the treatment ofrespiratory disorders." The smorgasbord of activities displayed byadenosine receptor agents underscores their great potential utility fortherapy and the need for central agents.

Adenosine exerts its various biological effects via action oncell-surface receptors. These adenosine receptors are of two types: A-1and A-2. The A-1 receptors are operationally defined as those receptorsat which several N6-substituted adenosine analogs such asR-phenylisopropyladenosine (R-PIA) and cycloadenosine (CHA) are morepotent than 2-chloroadenosine and N-5'-ethylcarboxamidoadenosine (NECA).At A-2 receptors the order of potency is insteadNECA>2-chloroadenosine>R-PIA>CHA.

As illustrated in the table above, adenosine receptors govern a varietyof physiological functions. The two major classes of adenosine receptorshave already been defined. These are the A-1 adenosine receptor, whichis inhibitory of adenylate cyclase, and the A-2 adenosine receptor,which is stimulatory to adenylate cyclase. The A-1 receptor has a higheraffinity for adenosine and adenosine analogs than the A-2 receptor. Thephysiological effects of adenosine and adenosine analogs are complicatedby the fact that non-selective adenosine receptor agents first bind therather ubiquitous low-affinity A-2 receptors, then as the dose isincreased, the high-affinity A-2 receptors are bound, and finally, atmuch higher doses, the very high-affinity A-1 adenosine receptors arebound. (See J. W. Daly, et al., Subclasses of Adenosine Receptors in theCentral Nervous System: Interaction with Caffeine and RelatedMethylxanthines, Cellular and Molecular Neurobiology, 3,(1), 69-80(1983).

In general, the physiological effects of adenosine are mediated byeither the stimulation or the inhibition of adenylate cyclase.Activation of adenylate cyclase increases the intracellularconcentration of cyclic AMP, which, in general, is recognized as anintracellular second messenger. The effects of adenosine analogs cantherefore be measured by either the ability to increase or the abilityto antagonize the increase in the cyclic AMP in cultured cell lines. Twoimportant cell lines in this regard are VA 13 (WI-38 VA 13 2RA), SV-40transformed WI 38 human fetal lung fibroblasts, which are known to carrythe A-2 subtype of adenosine receptor, and fat cells, which are known tocarry the A-1 subtype of adenosine receptor. (See R. F. Bruns, AdenosineAntagonism by Purines, Pteridines and Benzopteridines in HumanFibroblasts, Chemical Pharmacology, 30, 325-33 (1981).)

It is well known from in vitro studies that the carboxylic acid congenerof 8-phenyl-1,3-dipropyl-xanthine (XCC) is adenosine receptornonselective, with a Ki at the A-1 receptors in brain membranes of 58±3nM and a Ki at the A-2 receptors of the brain slice assay of 34±13 nM.The amino congener of 8-phenyl-1,3-dipropyl-xanthine (XAC), on the otherhand, has a 40-fold higher affinity for A-1 adenosine receptors, with aKi of 1.2±0.5 nM, as compared with a Ki at the A-2 receptors of thebrain slice assay of 49±17 nM. In addition, XAC is much more potent inantagonizing the effects of adenosine analogs on heart rate than onblood pressure. Since it is generally known that the adenosineanalog-induced effects on the heart seem to be mediated via A-1receptors and those on blood pressure via A-2 receptors, the selectivityof XAC under in vivo conditions suggests that adenosine receptoractivity in vitro correlates with adenosine receptor activity in vivoand that specific physiological effects can be distinguished as a resultof this selectivity. (See B. B. Fredholm, K. A. Jacobsen, B. Jonzon, K.L. Kirk, Y. O. Li, and J. W. Daly, Evidence That a Novel8-Phenyl-Substituted Xanthine Derivative is a Cardioselective AdenosineReceptor Antagonist In Vivo, Journal of Cardiovascular Pharmacology, 9,396-400(1987), and also K. A. Jacobsen, K. L. Kirk, J. W. Daly, B.Jonzon, Y. O. Li, and B. B. Fredholm, Novel 8-Phenyl-SubstitutedXanthine Derivative Is Selective Antagonist At Adenosine Receptors InVivo, Acta Physiol. Scand., 341-42 (1985).)

It is also known that adenosine produces a marked decrease in bloodpressure. This blood pressure reduction is probably dependent upon anA-2 receptor-mediated decrease in peripheral resistance. Adenosineanalogs are also able to decrease heart rate. This effect is probablymediated via adenosine receptors of the A-1 subtype.

Thus, it is readily apparent that the pharmacological administration ofthe adenosine receptor selective adenosine analogs disclosed herein willresult in selective binding to either the A-2 or the A-1 receptor, whichwill, in turn, selectively result in either a decrease in blood pressureor a decrease in heart rate, for example, thereby decoupling thesephysiological effects in vivo. The selection of such adenosine receptorselective agents can be determined by the methods described in furtherdetail below.

Test For Affinity For Brain Adenosine A-2 Receptors

The test described below was used to determine the potency of testcompounds to compete with the ligand[3H]-5'-N-ethyl-carboxamidoadenosine (NECA) for the adenosine A-2receptors prepared from animal brain membranes. (See also R. R. Bruns,G. H. Lu, and T. A. Pugsley, Characterization of the A-2 AdenosineReceptor Labeled by[3H]NECA in Rat Striatal Membranes, Mol. Pharmacol.,29, 331-346 (1986).) Young male rats (C-D strain), obtained from CharlesRiver, are killed by decapitation and the brains are removed. Membranesfor ligand binding are isolated from rat brain striatum. The tissue ishomogenized in 20 vol ice-cold 50 mM Tris-HCl buffer (pH 7.7) using apolytron (setting for 6 to 20 seconds). The homogenate is centrifuged at50,000×g for 10 minutes at 4° C. The pellet is again homogenized in apolytron in 20 vol of buffer, and centrifuged as before. The pellet isfinally resuspended in 40 vol of 50 mM Tris-HCl (pH 7.7 ) per gram oforiginal wet weight of tissue.

Incubation tubes, in triplicate, receive 100 μl of [3H]NECA (94 nM inthe assay), 100 μl of 1 μM cyclohexyladenosine (CHA), 100 μl of 100mMMgCl₂, 100 μl of 1 IU/ml adenosine deaminase, 100 μl of test compoundsat various concentrations over the range of 10⁻¹⁰ M to 10⁻⁴ M dilutedwith assay buffer (50 mM Tris-HCl, pH 7.7) and 0.2 μl of membranesuspension (5 mg wet weight), in a final volume of 1 ml of 50 mMTris-HCl, pH 7.7. Incubations are carried out at 25° C. for 60 minutes.Each tube is filtered through GF/B glass fiber filters using a vacuum.The filters are rinsed two times with 5 ml of the ice-cold buffer. Themembranes on the filters are transferred to scintillation vials to which8 ml of Omnifluor with 5% Protosol is added. The filters are counted byliquid scintillation spectrometry.

Specific binding of [3H]NECA is measured as the excess over blanks runin the presence of 100 μM 2-chloroadenosine. Total membrane-boundradioactivity is about 2.5% of that added to the test tubes. Since thiscondition limits total binding to less than 10% of the radioactivity,the concentration of free ligand does not change appreciably during thebinding assay. Specific binding to membranes is about 50% of the totalbound. Protein content of the membrane suspension is determined by themethod of O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall,Protein Measurements With Folin Phenol Reagent, J. Biol. Chem., 193,265-275 (1951).

Displacement of [3H]NECA binding of 15% or more by a test compound isindicative of affinity for the adenosine A-2 site. The molarconcentration of a compound which causes 50% inhibition of the bindingof ligand is the IC₅₀. A value in the range of 100-1000 nM wouldindicate a highly potent compound.

Test For Affinity For Brain Adenosine A-1 Receptor Binding Sites

The test described below is used to determine the potency of testcompounds to compete with the ligand [3H]-cycloadenosine for theAdenosine A-1 receptor prepared from rat brain membranes. MaleSprague-Dawley rats are sacrificed by decapitation and the membranes areisolated from whole animal brains. (See R. Goodman, M. Cooper, M.Gavish, and S. Synder, Guanine Nucleotide and Cation Regulation of theBinding of [3H]Diethyphenylxanthine to Adenosine A-1Receptors in BrainMembrane, Molecular Pharmacology, 21, 329-335 (1982).)

Membranes are homogenized (using polytron setting 7 for 10 seconds) in25 volumes of ice-cold 50 mM Tris-HCl buffer, pH 7.7. The homogenate iscentrifuged at 19,000 rpm for 10 minutes at 4° C. The pellet is washedby resuspending in 25 volumes of buffer with 2 IU of adenosine deaminaseper ml and incubated 30 minutes at 37° C. The homogenate is centrifugedagain. The final pellet is resuspended in 25 volumes of ice-cold buffer.

The incubation tubes, in triplicate, receive 100 μl of[3H]cyclohexyladenosine, 0.8 nM in the assay, 200 μl of test compoundsat various concentrations over the range of 10⁻¹⁰ M to 10⁻⁶ M dilutedwith 50 nM Tris-HCl buffer (pH 7.7), 0.2 ml of membrane suspension (8 mgwet weight) and in a final volume of 2 ml with Tris buffer. Incubationsare carried out at 25° C. for 2 hours and each one is terminated within10 seconds by filtration through a GF/B glass fiber filter using avacuum. The membranes on the filters are transferred to scintillationvials. The filters are counted by liquid scintillation spectrometry in 8ml of Omniflour containing 5% Protosol.

Specific binding of [3H]cycloadenosine is measured as the excess overblanks taken in the presence of 10⁻⁵ M 2-chloroadenosine. Totalmembrane-bound radioactivity is about 5% of that added to the testtubes. Specific binding to membranes is about 90% of the total bound.Protein content of the membrane suspension is determined by the methodof Lowry, et al. Ibid., 265.

Displacement of [3H]cyclohexyladenosine binding of 15% or more by a testcompound is indicative of affinity for the adenosine binding site.

Adenosine Receptor Binding Affinity Values Obtained Using The AboveDescribed Test Procedures

The following is a table showing the adenosine receptor bindingaffinities for several compounds (refer to compound examples on page 5for cross reference to compound names) within the scope of the presentinvention:

    ______________________________________                                                A-1         A-2                                                       Compound                                                                              Receptor Ki Receotor Ki  A-2 Ki/A-1 Ki                                ______________________________________                                        1.      7.40 × 10.sup.-6                                                                    6.38 × 10.sup.-5                                                                     11.80                                        2.      4.80 × 10.sup.-6                                                                    4.54 × 10.sup.-5                                                                     13.00                                        3.      1.10 × 10.sup.-7                                                                    5.90 × 10.sup.-6                                                                     72.20                                        4.      2.90 × 10.sup.-5                                                                    >1.99 × 10.sup.-4                                                                    --                                           5.      5.53 × 10.sup.-6                                                                    4.06 × 10.sup.-6                                                                     0.73                                         6.      6.43 × 10.sup.-7                                                                    1.31 × 10.sup.-6                                                                     2.04                                         7.      1.80 × 10.sup.-6                                                                    1.60 × 10.sup.-6                                                                     0.89                                         8.      2.01 × 10.sup.-6                                                                    3.63 × 10.sup.-6                                                                     1.80                                         9.      1.13 × 10.sup.-5                                                                    >6.99 × 10.sup.-6                                                                    --                                           10.     1.74 × 10.sup.-6                                                                    2.90 × 10.sup.-6                                                                     1.67                                         11.     3.21 × 10.sup.-7                                                                    3.77 × 10.sup.-7                                                                     1.17                                         12.     3.70 × 10.sup.-6                                                                    1.72 × 10.sup.-5                                                                     4.65                                         13.     4.40 × 10.sup.-8                                                                    1.90 × 10.sup.-6                                                                     43.18                                        14.      6.5 × 10.sup.-9                                                                    0.85 × 10.sup.-6                                                                     131.00                                       15.     0.66 × 10.sup.-6                                                                    14.4 × 10.sup.-6                                                                     22.00                                        ______________________________________                                    

The nucleotide guanosine triphosphate (GTP) has been shown todifferentially affect the binding of agonists and antagonists to avariety of neurotransmitter receptors. In general, guanine nucleotideslower the affinity of agonists for receptors without a concomitantdecrease in antagonist affinity. Accordingly, GTP has been shown todecrease the potency of agonists but not antagonists as inhibitors ofthe binding of the adenosine antagonist [3H]3-diethyl-8-phenylxanthine.In general, GTP greatly reduces the potency of purine agonists, but notantagonists as inhibitors of [3H]phenylisopropyl adenosine binding andis, therefore, an effective agent for distinguishing between agonistsand antagonists. (See L. P. Davies, S. C. Chow, J. H. Skerritt, D. J.Brown and G. A. R. Johnston, Pyrazolo[3,4-d]-Pyrimidines as AdenosineAntagonists, Life Sciences 34 2117-28 (1984).) It is understood, ingeneral, that adenosine analogs act as agonists if β-D-ribofuranosyl ispresent in the molecule at the R₁ position and as an antagonist if R₁ ishydrogen or phenyl.

Pharmaceutical Preparations of the Adenosine Receptor SelectionAdenosine Analogs

The exact amount of the compound or compounds to be employed, i.e., theamount of the subject compound or compounds sufficient to provide thedesired effect, depends on various factors such as the compoundemployed; type of administration; the size, age and species of animal;the route, time and frequency of administration; and, the physiologicaleffect desired. In particular cases, the amount to be administered canbe ascertained by conventional range finding techniques.

The compounds are preferably administered in the form of a compositioncomprising the compound in admixture with a pharmaceutically acceptablecarrier, i.e., a carrier which is chemically inert to the activecompound and which has no detrimental side effects or toxicity under theconditions of use. Such compositions can contain from about 0.1 μg orless to 500 mg of the active compound per ml of carrier to about 99% byweight of the active compound in combination with apharmaceutically-acceptable carrier.

The compositions can be in solid forms, such as tablets, capsules,granulations, feed mixes, feed supplements and concentrates, powders,granules or the like; as well as liquid forms such as sterile injectablesuspensions, orally administered suspensions or solutions. Thepharmaceutically acceptable carriers can include excipients such assurface active dispersing agents, suspending agents, tableting binders,lubricants, flavors and colorants. Suitable excipients are disclosed,for example, in texts such as Remington's Pharmaceutical Manufacturing,13 Ed., Mack Publishing Co., Easton, Pa. (1965).

The following examples are presented to illustrate the present inventionbut they should not be construed as limiting in any way.

EXAMPLE 1

To a solution of 2.5 g of 2,6-dichloropurine dissolved in 50 ml ethanolwas added 2.0 g (S)-(-)-2-amino-3-phenyl-1-propanol, and 1.83 ml Et₃ Nwith stirring at room temperature for 2 hours. The mixture was thenheated to reflux for 20 hours. The solvent was removed under vacuum andthe residue purified by flash chromatography (5-10% MeOH/CHCl₃) to yield3.68 g of a yellow solid,S-β-[(2-chloro-1H-purin-6-yl)amino]benzenepropanol (m.p. 117°-123° C).

This was followed by suspension of 1.42 g of the above product in 30 mlCHCl₃ and treatment with 1.30 g triphenylmethylchloride and 0.65 ml Et₃N. After 3 hours the reaction was diluted with 200 ml CHCl₃ and remixedwith 200 ml saturated NaHCO₃, 200 ml saturated NaCl, dried over MgSO₄,filtered and concentrated to yield 3.3 g of a yellow solid. This wasflash chromatographed (5% MeOH/CHCl₃) to yield 2.16 g of a foam product,S-β-[(9-triphenylmethyl-2-chloro-1H-purin-6-yl )amino]benzenepropanol.

To a solution of 330 mg sodium dissolved in 100 ml n-propanol was added2.15 g ofS-β-[(9-triphenylmethyl-2-chloro-1H-purin-6-yl)amino]benzenepropanol andthe reaction was heated to reflux for 6 hours. It was then cooled toroom temperature, poured into 300 ml H₂ O and extracted with CHCl₃ (3times 200 ml). The combined organic extracts were washed with 300 mlsaturated NaCl, dried over Na₂ SO₄, filtered and concentrated to yield2.16 g of a foam product,S-β-[(2-propoxy-9-triphenylmethyl-1H-purin-6-yl )amino]benzenepropanol.

Subsequently, 2.14 g ofS-β-[(2-propoxy-9-triphenylmethyl-1H-purin-6-yl)amino]benzenepropanolwas dissolved in 50 ml CH₂ Cl₂ followed by addition of p-toluenesulfonicacid (0.71 g). After stirring 24 hours the solvent was removed undervacuum and the residue was purified by flash chromatography (5-10%MeOH/CHCl₃) to yield 0.81 g of a white solid,(S)-β-[(2-propoxy-1H-purin-6-yl)amino]benzenepropanol (m.p. 229°-231°C.).

EXAMPLE 2

2.0 g of 6-chloro-9-phenylpurine was combined with 1.38 gR-(+)-2-amino-3-phenyl-1-propanol, 1.27 ml Et₃ N, 50 ml absolute ethanoland heated to reflux for 5 hours. The solvent was then removed and theresidue was purified by flash chromatography (5% MeOH/CHCl₃), followedby a second purification (2.5-5% MeOH/CHCl₃) to yield 2.66 g of a whitefoam (88% yield). This was recrystallized from 10% isopropylalcohol/hexane and dried under vacuum at 90° C. for four days to yield1.28 g of a white solid,(R)-β-[(9-phenyl-9H-purin-6-yl)amino]benzenepropanol (m.p. 130°-132°C.).

EXAMPLE 3

2.0 g of 6-chloro-9-phenylpurine was combined with 1.38 gS-(-)-2-amino-3-phenyl-1-propanol, 1.27 ml Et₃ N, 50 ml ethanol andheated to reflux for 5 hours. The solvent was then removed under vacuumand the residue purified by flash chromatography (2.5-5% MeOH/CHCl₃) toyield 2.27 g of product (76% yield). This was then recrystallized fromisopropyl alcohol/hexane 10% to yield after drying under vacuum at 90°C. for 3 days 0.87 g of a white solid,(S)-β-[(9-phenyl-9H-purin-6-yl)amino]benzenepropanol (m.p. 130°-132°C.).

EXAMPLE 4

1.94 g of D-amphetamine sulfate was made basic with 10% KOH. The aqueoussolution was extracted with ether. The organic layer was dried overMgSO₄, filtered, and concentrated to yield a clear oil. This was dilutedwith 3 ml of ethanol and added to a stirred solution of 466 g of1-phenyl-4,6-dichloropyrazolo[3,4-d]pyrimidine in 7 ml ethanol. After 48hours the solvent was removed under vacuum and the crude material waspurified by radial chromatography (20%-40% ethyl alcohol/hexane, 2 mmplate) to yield 640 mg of(S)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine(100%).

Next, 324 mg sodium was reacted with 10 ml of n-propanol. To this, 640mg of (S)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine in 5 ml ofn-propanol was added with stirring and the reaction was heated to 90° C.for 2 hours. It was then cooled, diluted with 200 ml saturated NaCl, andextracted with 200 ml CHCl₃. The organic layer was dried over MgSO₄,filtered, and concentrated to yield an oil which was purified by radialchromatography (30-50% Et₂ O/hexane) to yield after recrystallizationfrom 30% Et₂ O/hexane 382 mg of product (m.p. 134°-136° C.). Proton NMRindicated ether was still present. The compound was then oven driedunder vacuum for 6 hours to yield 318 mg of(S)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-amine(m.p. 134°-136° C.).

EXAMPLE 5

1.94 g of L-amphetamine sulfate was dissolved in H₂ O, made basic, andextracted with ether. The ether layer was concentrated under vacuum, theoil was taken up in 3 ml ethanol, and added to a stirred suspension of465 mg 1-phenyl-4,6-dichloropyrazolo[3,4-d]pyrimidine in 7 ml ethanol.After 24 hours the solvent was removed under vacuum and the crude oilwas purified by radial chromatography (40-60% Et₂ O/hexane, 2 mm plate)to yield 531 mg of(R)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine(83%).

Next, 268 mg sodium was reacted with 10 ml of n-propanol. 531 mg of(R)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-aminein 5 ml n-propanol was added to the stirred solution under nitrogen. Thereaction was heated (oil bath 90° C.) for 2 hours. It was then cooled,diluted with 100 ml saturated NaCl, and the aqueous solution wasextracted with 200 ml CHCl₃. The organic layer was then dried overMgSO₄, filtered, and concentrated to yield an oil which was purified byradial chromatography (30-50% Et₂ O/hexane, 2 mm plate) to yield, afterrecrystallization from 30% Et₂ O/hexane, 381.4 mg of a white solid (m.p.135°-137° C.). Proton NMR indicated ether was still present. Thus thecompound was oven dried under vacuum (setting at 3) for 6 hours to yield326 mg of final product,(R)-N-(1-methyl-2-phenylethyl)-1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-amine (m.p. 135°-137° C.).

EXAMPLE 6

1 g of 1-phenyl-4,6-dichlorpyrazolo[3,4-d]pyrimidine was suspended in 25ml ethanol. 1.71 g of 1R,2S-norephedrine was added with stirring. After24 hours the solvent was removed under vacuum and the crude oil waspurified by radial chromatography (40-50-60-70% Et₂ O/hexane, 4 mmplate) to yield 1.17 g of a white solid, [S-(R*,S*)]-α-[1-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanol(m.p. 164°-165° C. 82% yield)

Next, 194 mg sodium was reacted with 10 ml of n-propanol. 400 mg of[S-(R*,S*)]-α-[1-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanolin 5 ml of n-propanol was added to the stirred solution under nitrogen.The reaction was heated to 90° C. for 2 hours. It was then diluted with100 ml saturated NaCl, extracted with 200 ml CHCl₃, filtered, andconcentrated under vacuum to yield an oil which was purified by radialchromatography (5-10-20% isopropyl alcohol/hexane, 4 mm plate) to yield423 mg of an oil. Recrystallization from 20% Et₂ O/hexane and vacuumoven drying at 70° C. for 24 hours yield 122 mg of[S-(R*,S*)]-α-[1-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanol(m.p. 136°-138° C.).

EXAMPLE 7

390 mg of 1-phenyl-4,6-dichloropyrazolo[3,4-d]pyrimidine was suspendedin 15 ml 95% ethanol. 828 mg norephedrine HCl was dissolved in 100 ml H₂O, made basic with 10% KOH, and the free base extracted with 100 mlether. The organic was dried over MgSO₄, filtered, and concentrated toyield an oil which was added to the stirred reaction. After 4 hours thesolution became clear and the solvent was removed under vacuum. Thecrude oil was then purified by radial chromatography (5-10-20% isopropylalcohol/hexane, 4 mm plate) to yield 535 mg of[R-(S*,R*)]-α-[1-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanol(90%).

Next, 165 mg sodium was reacted with 10 ml of n-propanol. 341 mg of[R-(S*,R*)]-α-[1-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]ethyl]benzenemethanolin 3 ml of n-propanol was added with stirring and heated to 90° C. undernitrogen. After 2 hours the reaction was cooled and poured into 100 mlsaturated NaCl. It was then extracted with 200 ml CHCl₃, dried overMgSO₄, filtered, and concentrated to yield an oil which was purified byradial chromatography (5-10-20% isopropyl alcohol/hexane, 2 mm plate) toyield 326 mg product. This was recrystallized from 20% Et₂ O/hexane toyield 205 mg of a white solid,[R-(S*,R*)]-α-[1-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]-pyrimidin-4-yl)amino]ethyl]benzenemethanol(m.p. 137°-140° C.).

EXAMPLE 8

First, 2.5 g of 1-phenyl-4,6-dichloropyrazolo[3,4-d]-pyrimidine wassuspended in 60 ml ethanol, then 4.28 g (S)-(-)-2amino-3-phenyl-1-propanol was added and the reaction was allowed to stirfor 24 hours. The solvent was then removed under vacuum and the crudeoil was purified by flash chromatography (10-15-20% isopropylalcohol/hexane) to yield 3.5 g of product(S)-β-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanol(97%).

Next, 314 mg sodium was reacted with 15 ml of n-propanol. 650 mg of(S)-β-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanoldissolved in 10 ml of n-propanol was added to the reaction with stirringunder nitrogen. The reaction was then heated to 90° C. for two hours.After cooling it was poured into 100 ml saturated NaCl and extractedwith 200 ml CHCl₃. The organic layer was dried over MgSO₄, filtered, andconcentrated to yield an oil which was purified by radial chromatography(10-20% isopropyl alcohol/hexane) to yield after recrystallization from30% isopropyl alcohol/hexane and oven drying under vacuum at 60° C. for72 hours, 319 mg of(S)-β-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanol(46%, m.p. 155°-157° C.).

EXAMPLE 9

First, 2.81 g of 1-phenyl-4,6-dichloropyrazolo[3,4-d]pyrimidine wassuspended in 60 ml of ethanol, then 3.2 g of(R)-(+)-2-amino-3-phenyl-1-propanol was added with stirring. After 48hours the solvent was removed under vacuum and the oil was flashchromatographed (2-5-7% MeOH/CHCl₃) to yield 3.80 g of(R)-β-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanol(95%). Next, 380 mg of sodium was reacted with 10 ml of n-propanol. 773mg of(R)-β-[(1-phenyl-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanolin 5 ml of n-propanol was added with stirring. The reaction was heatedto 90° C. in an oil bath for 2.5 hours, then the solvent was removedunder vacuum, the residue taken up in 200 ml CHCl₃. The organic layerwas washed with saturated NaCl, dried over MgSO₄, filtered, andconcentrated to an oil which was purified by radial chromatography(10-20-30% isopropyl alcohol/hexane, 4 mm plate) to yield afterrecrystallization from 30% isopropyl alcohol/hexane 217 mg of a whitesolid (m.p. 158°-159° C.). Proton NMR indicated n-propanol was stillpresent, so this product was oven dried under vacuum (setting at 3) toyield 161 mg of final product,(R)-β-[(1-phenyl-6-propoxy-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-amino]benzenepropanol(m.p. 157°-158° C.).

EXAMPLE 10

First, 5 g of 2,6-dichloropurine and 8.4 g of ribose tetraacetate werecombined and heated to 155° C. with stirring to produce a heterogenoussuspension. A drop of concentrated H₂ SO₄ was added and the reaction wasallowed to stir at 155° C. until it became clear. The reaction wascooled and the HOAc was removed under vacuum. 30 ml of ethanol was addedand trituration, followed by filtration, yielded 3.7 g of product. Thiswas recrystallized from 175 ml ethanol to yield 2.31 g of2,6-dichloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine withmelting point of 154°-156° C. as long flat needles.

2.0 g of 2,6-dichloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-9H-purinewas combined with 0.67 g of S-(-)-2-amino-3-phenyl-1-propanol, 0.67 mltriethylamine, and heated to reflux for 16 hours. The solvent wasremoved and the residue was purified by flash chromatography (5-10%methanol/trichloromethane) to yield 0.90 g of a foam. Less purefractions were rechromatographed as above to provide a total of 1.81 gof(S)-β-[(9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-2-chloro-1H-purin-6-yl)amino]benzenepropanol.

Next, 0.59 g of sodium was reacted with 60 ml of n-propanol. Then-propoxide was then added to a stirring solution of 1.8 g of(S)-β-[(9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-2-chloro-1H-purin-6-yl)amino]benzenepropanolin 60 ml n-propanol and heated to reflux. After 16 hours, 3 ml water wasadded followed by MgSO₄. The reaction was filtered and concentratedunder vacuum. The residue was purified by flash chromatography (20-30%methanol/trichloromethane) to yield 840 mg of product. This wasrecrystallized from about 20% isopropyl alcohol/hexane and dried underhigh vacuum for 7 days to yield 197 mg of(S)-β-[(2-propoxy-9-(β-D-ribofuranosyl)-1H-purin-6-yl)amino]benzenepropanolas a white solid (m.p. 102°-104° C.).

EXAMPLE 11

First, 1.9 g of 2,6-dichloropurine and 3.2 g protected ribose(β-D-ribofuranose-1,2,3,5-tetraacetate) were combined and placed in anoil bath at 155° C. After 2 minutes of stirring a capillary drop ofconcentrated H₂ SO₄ was added and the reaction became homogeneous. Afterstirring an additional 10 minutes the reaction was cooled, the HOAc wasremoved under high vacuum and heat, and 15 ml of absolute ethanol added.The residue was dissolved with heat and the solution was placed in afreezer at -21° C. The white precipitate was collected andrecrystallized from 100 ml absolute ethanol to yield after drying undervacuum at 80° C. for 4 hours 2.16 g of2,6-dichloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-9H-purine having amelting point of 154°-157° C.

21 g of 2,6-dichloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-9H-purinewas combined with 0.7 g of R-(+)-2-amino-3-phenyl-1-propanol, 0.7 mltriethylamine and 75 ml absolute ethanol and heated to reflux for 4hours. The solvent was removed under vacuum and the residue purified byflash chromatography (5-10% methanol/trichloromethane ) to yield 2.27 gof (R)-β-[(9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-2-chloro-1H-purin-6-yl )amino]benzenepropanol.

Next, 0.72 g of sodium was reacted in 150 ml n-propanol. This solutionwas added to 2.2 g of(R)-β-[(9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-2-chloro-1H-purin-6-yl)amino]benzenepropanolwith stirring. The reaction was heated to reflux for 8 hours. Aftercooling the reaction was filtered and concentrated under vacuum. Theresidue was purified by flash chromatography (20-30%methanol/trichloromethane) to yield 930 mg of a foam product. This wasrecrystallized from about 10% isopropyl alcohol/hexane to yield afterdrying under vacuum at 80° C. for 24 hours 590 mg of(R)-β-[(2-propoxy-9-(β-D-ribofuranosyl)-1H-purin-6-yl)amino]benzenepropanolas a white solid. (m.p. 149°-152° C.).

EXAMPLE 12 ##STR7## Preparation of (R)-N-(1-phenylpropyl)-adenosine

Following the general procedure described by Schrecker (J. Org. Chem.,22, 33 (1957)), prepare the primary amine by dissolvingR-(-)-2-phenylbutyric acid (6.76 g, 41.2 mmol) in benzene (50 ml). Treatthe solution with oxalyl chloride (7.1 ml, 82.3 mmol) and slowly heatthe solution to 70° C. for 1 hour. After cooling, remove the solventunder vacuum at 38° C. Add benzene (2×50 ml) and again remove thesolvent under vacuum at 38° C. Dissolve the residue in acetone (25 ml)and add sodium azide (4 g in 12 ml water, 61.8 mmol) at 0° C. rapidlywith stirring. After 1 hour extract the reaction with benzene (2×150ml). Combine the organic extracts, dry over anhydrous magnesium sulfate,and filter. Slowly heat the filtrate to approximately 62° C. for 1 hour(nitrogen evolution). Cool the reaction, remove the solvent undervacuum, and add concentrated hydrochloric acid (22 ml) with stirring.Heat the reaction to 45° C. for 15 minutes. After cooling, add water(200 ml) and rinse the aqueous with diethyl ether (500 ml). Treat theaqueous solution cautiously with sodium hydroxide until it becomesbasic. Extract the aqueous with diethyl ether (4×150 ml), combine theorganic extracts, dry over anhydrous sodium sulfate, filter, andconcentrate under vacuum to provide the crude primary amine (4.53 g).Purify by flash chromatography (5 to 10% methanol/chloroform) to yieldR-(+)-1-phenylpropyl amine (3.52 g, [α]_(D) ²⁰ +15.4° C. (C=1.4,chloroform)).

Combine 6-chloropurine riboside (1 g, 3.49 mmol), theR-(+)-1-phenylpropyl amine (0.47 g, 3.49 mmol) prepared above,triethylamine (0.35 g, 3.49 mmol), and methanol (100 ml). Heat thereaction to reflux for 6 hours. After cooling, remove the solvent undervacuum and purify the residue by flash chromatography (5 to 10%methanol/chloroform) to provide 0.43 g of compound as a white foam. Dryover phosphorous pentoxide under high vacuum to yield the title compound(80 mg, m.p. 65°-74° C., [α]_(D) ²⁰ =-29.8° C. (C=1.06, chloroform)).

EXAMPLE 13 ##STR8## Preparation of (S)-N-(1-phenylpropyl)-adenosine

Following the general procedure described by Schrecker (Ibid), preparethe primary amine in a manner analogous to that described in Example 12from S-(+)-2-phenylbutyric acid (6 g, 36.5 mmol) to yieldS-(-)-1-phenylpropyl amine (2.75 g, [α]_(D) ²⁰ =-17.7° C. (C=0.976,chloroform)).

Combine 6-chloropurine riboside (1 g, 3.49 mmol), theS-(-)-1-phenylpropyl amine (0.47 g, 3.49 mmol) prepared above,triethylamine (0.35 g, 3.49 mmol), and methanol (50 ml). Heat thereaction to reflux for 7 hours. After cooling, remove the solvent undervacuum and purify the residue by flash chromatography (5 to 10%methanol/chloroform) to yield the title compound (0.72 g), m.p. 73°-84°C. [α]_(D) ²⁰ =-51.5° C. (C=1.00, chloroform).

What is claimed is:
 1. A compound according to the formula: ##STR9##wherein R₂ is hydrogen, lower alkyl of from 1 to 4 carbon atoms, orlower alkoxy of from 1 to 4 carbon atoms;X₁ and X₂ are eachindependently either hydrogen or hydroxy;with the proviso that X₁ and X₂cannot both be hydrogen.
 2. The compound which isβ-[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]benzenepropanol.